利用基因枪法将trxs基因导入大麦的研究

Study on Transgenic Barley Obtained by Biolistic Bombardment with trxs Gene

为提高大麦种子中硫氧还蛋白h的活力,用基因枪法将硫氧还蛋白基因家族中与trxh基因高度同源的trxs基因导入4550块大麦幼胚愈伤组织,经过愈伤组织的诱导、筛选(PPT)和分化,得到49株T0代再生植株,并进行繁殖得到T1、T2代植株。采用PCR、嵌套PCR、酶切PCR产物和PCR-southern杂交多种方法对获得的植物材料进行检测。PCR检测显示,T0及其后代T1、T2代植株中都检测出了PCR阳性个体;嵌套PCR和酶切检测验证了T1、T2代植株中PCR产物与转化的目的片断相符。对转基因大麦籽粒进行了初步的生化分析,结果显示转基因大麦籽粒中硫氧还蛋白h的活性是非转基因籽粒的3.3倍;发芽后水溶蛋白和醇溶蛋白中巯基含量的比率都明显上升,其中水溶蛋白质中巯基含量比率是对照的1.5倍,醇溶蛋白质中巯基含量比率是对照的2倍。以上结果说明trxs基因在转基因大麦中能进行遗传并具有生物功能。

To achieve barley with higher trx h activity and better malting qualities, trxs gene and bar gene (selective gene) were transformed into about 4550 barley immature embryos by biolistics bombardment . 49 regenerated plants were obtained via callus dedifferentiation induce, redifferentiation of shoot and root on selective medium containing PPT. The trxs gene in regenerated T_0 plants and their progeny as T_1 and T_2 plants were assayed by PCR, nested-PCR, enzymatic digestion and PCR-southern blotting. The results indicated that foreign trxs gene was integrated into the barley genomes integrallty and inherited to the progenies. Moreover, biochemistry analyses were carried out to discover the effects of trxs gene in transgenic seeds. The results showed that trx h activity in transgenic seeds was 3.3 times as great as that in non-transgenic seeds; Ratio of sulfhedryl in water soluble protein and alcohol soluble protein of barley seeds were enhanced to 1.5 fold and 2 fold respectively, which may advance proteins dissolution. Such results suggest that trxs gene in transgenic seeds can express its biofunction.